1 Induction and dorsal restriction of Paired-box 3 (Pax3) gene expression 2 in the caudal neuroectoderm is mediated by integration of multiple 3 pathways on a short neural crest enhancer 4 5 Oraly Sanchez-Ferras, Guillaume Bernas, Emilie Laberge-Perrault and Nicolas Pilon. 6 7 Molecular Genetics of Development Laboratory, Department of Biological Sciences and BioMed 8 Research Center, Faculty of Sciences, University of Quebec at Montreal (UQAM) 9 10 Running title: Regulation of Pax3 neural expression by a Cdx-Zic2 complex 11 12 To whom correspondence should be addressed: Nicolas Pilon, Department of Biological Sciences 13 and BioMed Research Center, Faculty of Sciences, University of Quebec at Montreal (UQAM), 141 14 President-Kennedy Ave, Montreal, PQ, Canada, H2X 3Y7; Tel.: 514-987-3000 x3342; Fax: 514-987- 15 4647; Email: [email protected] 16 17 18 ABSTRACT: 19 Pax3 encodes a paired-box transcription factor with key roles in neural crest and neural tube 20 ontogenesis. Robust control of Pax3 neural expression is ensured by two redundant sets of cis- 21 regulatory modules (CRMs) that integrate anterior-posterior (such as Wnt-Catenin signaling) as well 22 as dorsal-ventral (such as Shh-Gli signaling) instructive cues. In previous work, we sought to 23 characterize the Wnt-mediated regulation of Pax3 expression and identified the Cdx transcription 24 factors (Cdx1/2/4) as critical intermediates in this process. We identified the neural crest enhancer-2 25 (NCE2) from the 5’-flanking region of Pax3 as a Cdx-dependent CRM that recapitulates the restricted 26 expression of Pax3 in the mouse caudal neuroectoderm. While this is consistent with a key role in 27 relaying the inductive signal from posteriorizing Wnt ligands, the broad expression of Cdx proteins in 28 the tailbud region is not consistent with the restricted activity of NCE2. This implies that other positive 29 and/or negative inputs are required and, here, we report a novel role for the transcription factor Zic2 in 30 this regulation. Our data strongly suggests that Zic2 is involved in the induction (as a direct Pax3NCE2 31 activator and Cdx neural cofactor) as well as the maintenance of Pax3 dorsal restriction (as a target of 32 the ventral Shh repressive input). We also provide evidence that the inductive Cdx-Zic2 interaction is 33 integrated on NCE2 with a positive input from the neural-specific transcription factor Sox2. Altogether, 34 our data provide important mechanistic insights into the coordinated integration of different signaling 35 pathways on a short Pax3 CRM. 36 37 38 KEY WORDS: Cdx, Zic, Sox2, Pax3, neural crest cells, neural tube. 39 40 41 1 Regulation of Pax3 neural expression by a Cdx-Zic2 complex 42 1-INTRODUCTION 43 Building an embryo from an initial population of equivalent cells requires precise spatiotemporal 44 control of gene expression. Information to do this comes from just a few numbers of conserved 45 signaling pathways, is transmitted by DNA binding proteins and interpreted at the cis-regulatory level 46 on evolutionarily conserved genomic sequences. Redundant and different operating enhancers may 47 exist to refine and protect expression of developmental genes from fluctuations in these signals or 48 mutations in the genome. Subsequently, the output of gene expression and gene regulatory interactions 49 provide the memory to maintain established expression patterns in the absence of signaling inputs. In 50 this regard, a lot of work has been done to understand the molecular mechanisms of neural gene 51 expression during establishment of the anterior-posterior (AP) as well as the dorsal-ventral (DV) axes. 52 Nevertheless, how both AP and DV signaling inputs are coordinately integrated at the cis-regulatory 53 level is still poorly understood. 54 55 Pax3/7 (Paired box 3 and 7) and Zic (Zinc finger protein of the cerebellum) family members (Zic1-5) 56 encode transcription factors that exhibit overlapping expression domains in the neuroectoderm along 57 both the AP and the DV axis. During neurulation, expression of these genes is similarly restricted to 58 pre-migratory neural crest cells (NCC) and dorsal neural tube (NT) [1-9]. However, such overlap is less 59 extensive along the AP axis and most especially in the caudal embryo where only Pax3 and the 60 Zic2/Zic5 gene pair are expressed in the posterior neural plate (PNP) [1, 10]. Consistent with their wide 61 expression pattern and key developmental role, loss-of-function mutations of Pax3 and Zic2 – as seen 62 for example in the Splotch (Pax3) and Kumba (Zic2) mouse mutants – causes severe and similar NT 63 and NCC defects affecting the entire AP axis such as spina bifida, craniofacial malformations, absence 64 of dorsal root ganglia and pigmentary anomalies [6, 7, 10-12]. 65 66 Work performed in several vertebrate species has revealed that AP instructive cues from Wnt 67 (Wingless and Int-1 related) and FGF (fibroblast growth factor) pathways as well as DV instructive 68 cues from BMP (bone morphogenetic protein) and Shh (Sonic Hedgehog) pathways are all involved in 69 the induction and dorsal restriction of Pax3/7 and Zic members [8, 13-22]. These studies notably point 70 to a critical role for posteriorizing canonical Wnt signaling and intermediate levels of BMP molecules 71 during induction in the neural plate [14, 16-18, 23, 24] whereas opposing gradients of dorsal BMP and 72 ventral Shh signaling are subsequently implicated in the maintenance and dorsal restriction in the 73 closed NT [19-21, 25]. Although the general role of these pathways is well accepted, some species- 74 specific variations are also expected regarding their relative importance. In the case of Pax3, this is 75 well exemplified by the comparison of its posterior expression domain between chick and mouse 76 embryos. Indeed, in chick embryos, Pax3 expression is initially induced in the whole PNP before 77 becoming restricted to the dorsal NT whereas in mouse embryos, Pax3 expression is already restricted 78 to the lateral borders of the PNP during the induction phase [15, 26]. 79 80 Multiple evolutionary conserved cis-regulatory modules (CRMs) have been identified for Pax3. These 81 CRMs are clustered in two areas of the Pax3 locus: one in the 5’-flanking region and the other in 82 intron-4 [27-29]. The 5’-flanking region, named Neural Crest Enhancer (NCE), is located within the 2 Regulation of Pax3 neural expression by a Cdx-Zic2 complex 83 1.6 Kb proximal promoter and is subdivided in two short CRMs of approximately 250 bp named NCE1 84 and NCE2 [27, 28]. The entire NCE is not only able to direct Pax3 reporter expression in mouse NCC 85 and dorsal NT along the hindbrain and trunk region, but also drive enough functional expression levels 86 of Pax3 to rescue the NT and NCC defects observed in Pax3 Splotch mice [7]. Interestingly, targeted 87 deletion studies in the mouse have suggested that NCE acts redundantly with a second evolutionary 88 conserved region (ECR2) located in the 4th intron [29]. In fact, more recent work using the zebrafish as 89 a model has demonstrated that the Pax3 intron-4 contains at least two CRMs that appears to exhibit 90 complementary activities in order to recapitulate the induction, dorsal restriction and maintenance of 91 Pax3 neural expression [18, 30]. Given that the effect of their deletion has not been documented so far, 92 the requirement of any of these intron-4 CRMs for Pax3 expression as well as their relative importance 93 over the 5’-flanking NCE is currently unknown. 94 95 We have previously demonstrated that the NCE2 region alone is able to recapitulate both the induction 96 and dorsal restriction of Pax3 expression in the caudal NCC and NT [26], suggesting that this CRM is 97 well suited for analyzing the coordinated integration of both AP and DV instructive cues. In this regard, 98 we have already demonstrated that activity of this enhancer depends on a positive input from caudal- 99 related homeobox (Cdx) transcription factors downstream of Wnt/Catenin signaling [26]. Here we 100 further show that, in addition to Cdx, Zic2 also directly regulates murine Pax3 expression and acts as a 101 Cdx neural cofactor. Importantly; we show that the NCE2 region integrates positive inputs from caudal 102 Cdx, dorsal Zic2 as well as neural Sox2 transcription factors. Furthermore, we provide evidence for a 103 putative role of Zic2/5 as mediators of the Shh-induced repressive input involved in the dorsal 104 restriction of Pax3 expression. Taken together with previous descriptions of other functional binding 105 sites (e.g. Brn1 and Tead2) within NCE2 [28, 31], our data strongly suggest that this short CRM 106 behaves as a “super-enhancer” [32] that mediates the spatiotemporal induction and dorsal restriction of 107 Pax3 expression in the mouse caudal neuroectoderm. 108 109 2-MATERIALS and METHODS 110 111 2.1-Ethics Statement 112 Experiments involving mice were performed following Canadian Council of Animal Care (CCAC) 113 guidelines for the care and manipulation of animals used in medical research. Protocols involving the 114 manipulation of animals were approved by the institutional ethics committee of the University of 115 Quebec at Montreal (comité institutionnel de protection des animaux (CIPA)); Reference number 0513- 116 C1-648-0514). 117 118 2.2-Plasmid constructs and site-directed mutagenesis 119 The Pax3 cDNA vector pBH3.2 [1] was kindly provided by J. Epstein. Expression vectors for FLAG- 120 tagged Cdx1 and GST-Cdx1 fusion proteins have been described previously [33]. Expression vectors 121 for FLAG-tagged full-length and deletion mutant ZIC2 proteins were a generous gift from S. Tejpar 122 [34].